It is the overall aim of the current proposal to employ a combined spectroscopy/imaging approach using IR and Raman spectroscopy as well as fluorescence microscopy for the study of biophysical monolayer models of pulmonary surfactant. A combined approach is needed, since no one method is able to accomplish both structural characterization and morphological imaging for a detailed understanding of conformational and spatial properties in the monolayer. The combination of vibrational spectroscopy and fluorescence imaging will correlate molecular properties with mesoscopic (i.e. mum size) domain morphology in surfactant monolayers. Our recent results suggest that the interaction of surfactant proteins with biomembrane interfaces may occur selectively at locations with distinct morphological properties, such as at domain boundaries or areas of a certain lipid phase. Therefore, investigation of the molecular structural properties of the system, as reported by IR or Raman, in conjunction with its morphological structure, as visualized by fluorescence, is needed to more fully understand the interaction between the system components. The combination of techniques described in this proposal will mark an important achievement for the investigation of the molecular interaction between proteins and lipids in surfactant models in that we will be able to associate specific molecular spectroscopic properties with lateral organization in surface monolayers. During the next grant period, we will address the following specific aims: 1) to characterize the interfacial and hydrophobic interactions of the surfactant proteins SP-B and SP-C on monolayer structure by IR and to determine protein structure in the monolayer by Raman spectroscopy, 2) to characterize the influence of SP-B and SP-C on monolayer morphology by fluorescence microscopy and to determine how these proteins produce three-dimensional, surface-excluded material as seen using scattered light microscopy, 3) to use IR spectroscopy to measure microphase separation and domain formation in binary mixtures, and 4) to use spatially-resolved Raman microscopy to study lateral organization and molecular structure in surfactant monolayer films.